Many medical diagnosis processes are based on the detection of biomarker molecules such as proteins, antibodies, enzymes, DNA or RNA that are often uniquely associated with a particular disease. When diseases such as cancer appear in a human body, the human body is known to produce levels of certain chemicals that are much lower, or non-existent, in a healthy human being. For example, the diagnosis of prostate cancer is initially based on the concentration of a protein called PSA in the body. When the concentration of PSA exceeds a certain normal range, the doctor will generally suggest other more direct tools such as biopsy and imaging techniques to confirm the diagnosis of prostate cancer. Another example is breast cancer. Breast cancer patients are known to have much higher level of a certain enzyme called carbonic anhydrase. The detection of an unusually high level of this enzyme can provide the initial tool for breast cancer diagnosis.
The accurate detection of biomolecules at extremely low levels is vital for early diagnosis of diseases. Regarding many cancers and other diseases, if diagnosed early, the chance for successful cure or treatment is much higher than being diagnosed at later stages. Enormous research efforts have been and are continuously being pursued toward techniques and tools for biomolecule detection at ultra low levels. For example, enzymes, fluorescent dye molecules, and radioactive isotopes have been used extensively for bioconjugation and bioassays. Among the different biolabels, fluorescent dye molecules have received much attention due to the high sensitivity common to fluorescence detection. Although fluorescent dye molecules display shortcomings such as photobleaching, instability, and sensitivity to environmental conditions such as pH variation, some of these problems are being overcome by introducing highly luminescent and photostable quantum dots and nanoparticles. Quantum dots have high quantum yields, high molar extinction coefficients, high resistance to photobleaching and exceptional resistance to photo- and chemical degradation. Due to these exceptional optical properties, quantum dots have become one of the most interesting materials for bioimaging, labeling, and sensing. Other types of nanoparticle materials, such as gold and silver, exhibit some other unique size-dependent optical properties such as surface plasmon resonance (SPR). The extinction coefficient of metal nanoparticles is orders of magnitude higher than typical organic molecules; therefore, low concentration detection of DNAs based on color change of gold nanoparticle-DNA probe conjugates has been developed. Another important optical property of metal nanoparticles, the surface enhanced Raman scattering, is also being studied for ultra low level detection of biomolecules.
In addition to the development of labeling materials that can lead to lower detection limit, new techniques and methodologies to concentrate the analyte molecules and/or amplify the analyte concentration have also been reported. An example of this approach is the barcode detection of proteins and DNAs using gold nanoparticles and magnetic microparticles developed by Mirkin et al. (Nam, J; Park, S.; Mirkin, C. A. “Bio-barcodes based on oligonucleotide-modified nanoparticles” J. Am. Chem. Soc. 2002, 124, 3820) The magnetic microparticles are used as a tool to concentrate analyte molecules in solution by applying magnetic field. To detect the analyte molecules, multiple bar code DNA molecules are attached to the gold nanoparticle that is conjugated to the detector molecule. The detection of the analyte molecule is realized indirectly by measuring the amount of the bar code DNA molecules attached to gold nanoparticles. To increase the detection limit, the concentration of DNA barcode can be increased by PCR amplification. Using the bio-barcode method, Mirkin et al. has achieved detection limits for DNA molecules at the attomolar range (10−18 M) or lower. Similar approach has been demonstrated to detect prostate specific antigens (PSA) at attomolar concentration. Another extensively explored research area for protein detection is the use of DNA aptamers and PCR amplification technique. Specific binding towards a target protein is created by simultaneous binding of two DNA aptamers to two different sites of the same protein. Ligation of the two-closely positioned DNA aptamers followed by PCR amplification can lead to detection of target proteins at a level as low as zeptomole (10−21) range.
Despite significant progress, there is a strong and urgent need to develop more sensitive, reliable and low cost techniques for biomolecular detection and analysis at ultra low level. Although the bar-code method developed by Mirkin et al. has pushed the detection limit beyond the attomolar range, this method involves the use of expensive biomolecules (DNA) and rather sophisticated procedures and analytical instrumentation. Moreover, the amplification effect of the bar-code method is limited to the number of DNA barcodes that can be attached to the nanoparticle surface.
Regarding the fluorescence detection technique, the fluorescence of organic dyes and quantum dots is often affected by the chemical environment of the sample solution, and such effect could be further manifested at ultra low concentration. Although surface enhanced Raman scattering has shown promising potential for label-free trace detection of biomolecules, the Raman enhancement effect is not well understood and further study is needed before a routine assay method can be developed from this effect. For the DNA aptamer ligation method, the biggest problem is that PCR amplification is needed for the analysis and the type of proteins that can be detected using this method is relatively limited.
Therefore, a facile and economic method that allows the detection of a wide range of biomarker and other biologically significant target molecules at ultra low concentration is needed preferably in a quantitative manner.